Carrier nanoparticles and related compositions, methods and systems

ABSTRACT

Carrier nanoparticles comprising a polymer containing a polyol coupled to a polymer containing a boronic acid, configured to present the polymer containing a boronic acid to an environment external to the nanoparticle and related compositions, methods and systems.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 12/540,319, filed on Aug. 12, 2009, which claimspriority to U.S. provisional application Ser. No. 61/188,855, filed onAug. 13, 2008, each of these applications is incorporated by referenceherein.

GOVERNMENT RIGHTS

This invention was made with government support under CA 119347 awardedby the National Cancer Institute of the United States Department ofHealth and Human Services. The government has certain rights in theinvention.

FIELD

The present disclosure relates to carrier nanoparticles and inparticular to nanoparticles suitable for delivering compounds ofinterest, and related compositions, methods and systems.

BACKGROUND

Effective delivery of compounds of interest to cells, tissues, organs,and organisms has been a challenge in biomedicine, imaging and otherfields where delivery of molecules of various sizes and dimensions to apredetermined target is desirable.

Whether for pathological examination, therapeutic treatment or forfundamental biology studies, several methods are known and used fordelivering various classes of biomaterials and biomolecules which aretypically associated with a biological and/or chemical activity ofinterest.

As the number of molecules suitable to be used as chemical or biologicalagents (e.g. drugs, biologics, therapeutic or imaging agents) increases,development of a delivery systems suitable to be used with compounds ofvarious complexity, dimensions and chemical nature has proven to beparticularly challenging.

Nanoparticles are structures useful as carriers for delivering agentswith various methods of delivery. Several nanoparticle delivery systemsexist, which utilize an array of different strategies to package,transport, and deliver an agent to specific targets.

SUMMARY

Provided herein are nanoparticles and related compositions, methods andsystems that in several embodiments provide a multifunctional tool foreffective and specific delivery of a compound of interest. Inparticular, in several embodiments, nanoparticles herein described canbe used as a flexible system for carrying and delivering a wide range ofmolecules of various sizes, dimensions and chemical nature topredetermined targets.

According to a first aspect, a nanoparticle comprising a polymercontaining a polyol and to a polymer containing a boronic acid isdescribed. In the nanoparticle, the polymer containing a boronic acid iscoupled to the polymer containing a polyol and the nanoparticle isconfigured to present the polymer containing a boronic acid to anenvironment external to the nanoparticle. In the nanoparticle one ormore compounds of interest can be carried by the nanoparticle, as a partof or attached to the polymer containing a polyol and/or the polymercontaining a boronic acid.

According to a second aspect, a composition is described. Thecomposition comprises a nanoparticle herein described and a suitablevehicle and/or excipient.

According to a third aspect, a method to deliver a compound to a targetis described. The method comprises contacting the target with ananoparticle herein described wherein the compound is comprised in thepolymer containing a polyol or in the polymer containing a boronic acidof the nanoparticle herein described.

According to a fourth aspect, a system to deliver a compound to a targetis described. The system comprises at least a polymer containing apolyol and polymer containing a boronic acid capable of reciprocalbinding through a reversible covalent linkage, to be assembled in ananoparticle herein described comprising the compound.

According to a fifth aspect, a method to administer a compound to anindividual is described. The method comprises administering to theindividual an effective amount of a nanoparticle herein described,wherein the compound is comprised in the polymer containing a polyoland/or in the polymer containing a boronic acid.

According to a sixth aspect, a system for administering a compound to anindividual is described. The system comprises, at least a polymercontaining a polyol and polymer containing a boronic acid capable ofreciprocal binding through a reversible covalent linkage, to beassembled in a nanoparticle herein described attaching the compound tobe administered to the individual according to methods herein described.

According to a seventh aspect, a method to prepare a nanoparticlecomprising a polymer containing a polyol and a polymer containing aboronic acid is described. The method comprises contacting the polymercontaining polyols with the polymer containing a boronic acid for a timeand under condition to allow coupling of the polymer containing polyolywith the polymer containing a boronic acid.

According to an eight aspect, several polymer containing a boronic acidare described which are illustrated in details in the following sectionsof the present disclosure.

According to a ninth aspect, several polymers containing polyols aredescribed, which are illustrated in details in the following sections ofthe present disclosure.

Nanoparticles herein described and related compositions, methods, andsystems can be used in several embodiments as a flexible molecularstructure suitable for carrying compounds of various sizes, dimensionsand chemical nature.

Nanoparticles herein described and related compositions, methods, andsystems can be used in several embodiments as delivery systems which canprovide protection of the carried compound from degradation, recognitionby immune system and loss due to combination with serum proteins orblood cells.

Nanoparticles herein described and related compositions, methods, andsystems can be used in several embodiments as delivery systemscharacterized by steric stabilization and/or ability to deliver thecompound to specific targets such as tissues, specific cell types withina tissue and even specific intracellular locations within certain celltypes.

Nanoparticles herein described and related compositions, methods, andsystems can be designed in several embodiments, to release a carriedcompound in a controllable way, including controlled release of multiplecompounds within a same nanoparticle at different rates and/or times.

Nanoparticles herein described and related compositions, methods, andsystems can be used in several embodiments, to deliver compounds withenhanced specificity and/or selectivity during targeting and/or enhancedrecognition of the compound by the target compared to certain systems ofthe art.

Nanoparticles herein described and related compositions, methods, andsystems can be used in several embodiments in connection withapplications wherein controlled delivery of a compound of interest isdesirable, including but not limited to medical applications, such astherapeutics, diagnostics and clinical applications. Additionalapplications comprise biological analysis, veterinary applications, anddelivery of compounds of interest in organisms other than animals, andin particular in plants.

The details of one or more embodiments of the disclosure are set forthin the accompanying drawings and the detailed description and examplesbelow. Other features, objects, and advantages will be apparent from thedetailed description, examples and drawings, and from the appendedclaims

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and constitute apart of this specification, illustrate one or more embodiments of thepresent disclosure and, together with the detailed description and theexamples, serve to explain the principles and implementations of thedisclosure.

FIG. 1 shows a schematic representation of a nanoparticle and a relatedmethod for the relevant formation in absence of a boronic acidcontaining compound. Panel A shows a schematic representation of apolymer containing a polyol (MAP, 4) and a compound of interest (nucleicacid) according to an embodiment herein described. Panel B shows ananoparticle formed upon assembly of the polymer containing a polyol andcompound shown in panel A.

FIG. 2 shows a schematic representation of a nanoparticle and a relatedmethod of manufacturing according to an embodiment of the presentdisclosure. Panel A shows a polymer containing a polyol (MAP, 4) and apolymer containing a boronic acid (BA-PEG, 6) together with a moleculeof interest (nucleic acid) according to an embodiment of the presentdisclosure. Panel B shows a BA-pegylated stabilized nanoparticle formedupon assembly of the polymers and compound shown in panel A.

FIG. 3 shows formation of a complex comprising polymers containingpolyols and a compound of interest according to an embodiment hereindescribed. In particular, FIG. 3, shows results of a MAP gel retardationassay with plasmid DNA according to an embodiment of the presentdisclosure. A DNA ladder is loaded in Lane 1. Lanes 2-8 show plasmid DNAcombined with MAP of incrementally increased charge ratio. Charge ratiois defined as the amount of positive charges on the MAP divided by theamount of negative charges on the nucleic acid.

FIG. 4 shows formation of a complex comprising polymers containingpolyols and a compound of interest according to an embodiment hereindescribed. In particular, FIG. 4 shows results of a MAP gel retardationassay with siRNA according to an embodiment of the present disclosure. ADNA ladder is loaded in Lane 1. Lanes 2-8 show siRNA combined with MAPof incrementally increased charge ratio.

FIG. 5 shows properties of nanoparticles according to some embodimentsherein described. In particular, FIG. 5 shows a diagram illustrating aplot of particle size (determined from dynamic light scattering (DLS)measurements) versus charge ratio and zeta potential (a property thatrelates to the surface charge of the nanoparticle) versus charge ratiofor MAP-plasmid nanoparticles according to an embodiment of the presentdisclosure.

FIG. 6 properties of nanoparticles according to some embodiments hereindescribed. In particular, FIG. 6 shows a diagram illustrating a plot ofparticle size (DLS) versus charge ratio and zeta potential versus chargeratio for BA-PEGylated MAP-plasmid nanoparticles according to anembodiment of the present disclosure.

FIG. 7 shows the salt stability of BA-PEGylated MAP-PlasmidNanoparticles according to an embodiment herein disclosed. Plot A: 5:1BA-PEG+np+1×PBS after 5 mins; Plot B: 5:1 BA-PEG+np, dialyzed 3×w/100kDa+1×PBS after 5 mins; Plot C: 5:1 prePEGylated w/BA-PEG+1×PBS after 5mins; Plot D: 5:1 prePEGylated w/BA-PEG, dialyze 3×w/100 kDa+PBS after 5mins.

FIG. 8 shows delivery of an agent to human cells in vitro withnanoparticles according to an embodiment herein described. Inparticular, FIG. 8 shows a diagram illustrating a plot of relative lightunits (RLU) that are a measure of the amount of luciferase proteinexpressed from the pGL3 plasmid that has been delivered to the cellsversus charge ratio for a MAP/pGL3 transfection into HeLa Cellsaccording to an embodiment of the present disclosure.

FIG. 9 shows delivery of an agent to a target with nanoparticlesaccording to an embodiment herein described. In particular, FIG. 9,shows a diagram illustrating a plot of cell survival versus charge ratioafter a MAP/pGL3 transfection according to an embodiment of the presentdisclosure. The survival data are for the experiments shown in FIG. 8.

FIG. 10 shows delivery of multiple compounds to a target withnanoparticles according to an embodiment herein described. Inparticular, FIG. 10 shows a diagram illustrating a plot of relativelight units (RLU) versus particle type for a co-transfection of MAPParticles containing pGL3 and siGL3 at a charge ratio of 5+/− into HeLaCells according to an embodiment herein described. The wording siCONindicates an siRNA with a control sequence.

FIG. 11 shows delivery of a compound to a target with nanoparticlesaccording to an embodiment herein described. In particular, FIG. 11shows a plot of relative light units (RLU) versus siGL3 concentrationfor a delivery of MAP/siGL3 at a charge ratio of 5+/− into HeLa-Luccells according to an embodiment of the present disclosure.

FIG. 12 shows a schematic representation of a synthesis of a polymercontaining a boronic acid presenting a targeting ligand according tosome embodiments herein described. In particular FIG. 12, show aschematic for a synthesis of boronic acid-PEG disulfide-Transferrinaccording to an embodiment of the present disclosure.

FIG. 13 shows a schematic representation of a synthesis of ananoparticle according to some embodiments herein described. Inparticular, FIG. 13 shows a schematic for a formulation of ananoparticle with Campothecin Mucic acid polymer (CPT-mucic acidpolymer) in water according to an embodiment of the present disclosure.

FIG. 14 shows a table summarizing particle sizes and zeta potentials ofnanoparticles formed from the CPT-mucic acid polymer conjugated inwater, prepared according to an embodiment of the present disclosure.

FIG. 15 shows a schematic representation of a synthesis of ananoparticle according to some embodiments herein described. Inparticular, FIG. 15 shows a formulation of a boronic acid-PEGylatednanoparticle with CPT-Mucic Acid Polymer and boronicacid-disulfide-PEG₅₀₀₀ in water according to an embodiment of thepresent disclosure.

DETAILED DESCRIPTION

Provided herein are nanoparticles and related compositions, methods, andsystems that can be used in connection for delivering a compound ofinterest (herein also cargo) comprised in the nanoparticles.

The term “nanoparticle” as used herein indicates a composite structureof nanoscale dimensions. In particular, nanoparticles are typicallyparticles of a size in the range of from about 1 to about 1000 nm, andare usually spherical although different morphologies are possibledepending on the nanoparticle composition. The portion of thenanoparticle contacting an environment external to the nanoparticle isgenerally identified as the surface of the nanoparticle. Innanoparticles herein described, the size limitation can be restricted totwo dimensions and so that nanoparticles herein described includecomposite structure having a diameter from about 1 to about 1000 nm,where the specific diameter depends on the nanoparticle composition andon the intended use of the nanoparticle according to the experimentaldesign. For example, nanoparticles to be used in several therapeuticapplications typically have a size of about 200 nm or below, and theones used, in particular, for delivery associated to cancer treatmenttypically have a diameter from about 1 to about 100 nm.

Additional desirable properties of the nanoparticle, such as surfacecharges and steric stabilization, can also vary in view of the specificapplication of interest. Exemplary properties that can be desirable inclinical applications such as cancer treatment are described in Davis etal, 2008, Duncan 2006 and Allen 2002 each incorporated herein byreference in its entirety. Additional properties are identifiable by askilled person upon reading of the present disclosure. Nanoparticledimensions and properties can be detected by techniques known in theart. Exemplary techniques to detect particles dimensions include but arenot limited to dynamic light scattering (DLS) and a variety ofmicroscopies such at transmission electron microscopy (TEM) and atomicforce microscopy (AFM). Exemplary techniques to detect particlemorphology include but are not limited to TEM and AFM. Exemplarytechniques to detect surface charges of the nanoparticle include but arenot limited to zeta potential method. Additional techniques suitable todetect other chemical properties comprise by ¹H, ¹¹B, and ¹³C and ¹⁹FNMR, UV/Vis and infrared/Raman spectroscopies and fluorescencespectroscopy (when nanoparticle is used in combination with fluorescentlabels) and additional techniques identifiable by a skilled person.

Nanoparticles and related compositions, methods, and systems hereindescribed can be used to deliver a compound of interest and inparticular an agent to a predetermined target.

The term “deliver” and “delivery” as used herein indicates the activityof affecting the spatial location of a compound, and in particularcontrolling said location. Accordingly, delivering a compound in thesense of the present disclosure indicates the ability to affectpositioning and movement of the compound at a certain time under acertain set of conditions, so that the compound's positioning andmovement under those conditions are altered with respect to thepositioning and movement that the compound would otherwise have.

In particular, delivery of a compound with respect to a referenceendpoint indicates the ability to control positioning and movement ofthe compound so that the compound is eventually positioned on theselected reference endpoint. In an in vitro system, delivery of acompound is usually associated to a corresponding modification of thechemical and/or biological detectable properties and activities of thecompound. In an in vivo system, delivery of a compound is also typicallyassociated with modification of the pharmacokinetics and possiblypharmacodynamics of the compound.

Pharmacokinetic of a compound indicates absorption, distribution,metabolism and excretion of the compound from the system, typicallyprovided by the body of an individual. In particular the term“absorption” indicates the process of a substance entering the body, theterm “distribution” indicates the dispersion or dissemination ofsubstances throughout the fluids and tissues of the body, the term“metabolism” indicates the irreversible transformation of parentcompounds into daughter metabolites and the term “excretion” indicatesthe elimination of the substances from the body. If the compound is in aformulation, pharmacokinetics also comprises liberation of the compoundfrom the formulation which indicates process of release of the compound,typically a drug, from the formulation. The term “pharmacodynamic”indicates physiological effects of a compound on the body or onmicroorganisms or parasites within or on the body and the mechanisms ofdrug action and the relationship between drug concentration and effect.A skilled person will be able to identify the techniques and proceduressuitable to detect pharmacokinetics and pharmacodynamic features andproperties of a compound of interest and in particular of an agent ofinterest such as a drug.

The term “agent” as used herein indicates a compound capable ofexhibiting a chemical or biological activity associated to the target.The term “chemical activity” as used herein indicates the ability of themolecule to perform a chemical reaction. The term biological activity asused herein indicates the ability of the molecule to affect a livingmatter. Exemplary chemical activities of agents comprise formation of acovalent or electrostatic interaction. Exemplary biological activitiesof agents comprise production and secretion of endogenous molecules,absorption and metabolization of endogenous or exogenous molecules andactivation or deactivation of genetic expression including transcriptionand translation of a gene of interest.

The term “target” as used herein indicates a biological system ofinterest including unicellular or pluricellular living organisms or anyportion thereof and include in vitro or in vivo biological systems orany portion thereof.

The nanoparticles herein described a polymer containing a boronic acidsis coupled to a polymer containing a polyol is arranged in thenanoparticle to be presented to an environment external to thenanoparticle.

The term a “polymer” as used herein indicates a large molecule composedof repeating structural units typically connected by covalent chemicalbonds. A suitable polymer may be a linear and/or branched, and can takethe form of a homopolymer or a co-polymer. If a co-polymer is used, theco-polymer may be a random copolymer or a branched co-polymer. Exemplarypolymers comprise water-dispersible and in particular water solublepolymers. For example, suitable polymers include, but are not limited topolysaccharides, polyesters, polyamides, polyethers, polycarbonates,polyacrylates, etc. For therapeutic and/or pharmaceutical uses andapplications, the polymer should have a low toxicity profile and inparticular that are not toxic or cytotoxic. Suitable polymers includepolymers having a molecular weight of about 500,000 or below. Inparticular, suitable polymers can have a molecular weight of about100,000 and below.

The term “polymer containing a polyol” or “polyol(s) polymer” as usedherein indicates a polymer presenting multiple hydroxyl functionalgroups. In particular, the polymer containing a polyol suitable to formthe nanoparticles here described comprise polymers presenting at least aportion of the hydroxyl functional groups for a coupling interactionwith at least one boronic acid of a polymer containing a boronic acid.

The term “present” as used herein with reference to a compound orfunctional group indicates attachment performed to maintain the chemicalreactivity of the compound or functional group as attached. Accordingly,a functional group presented on a surface, is able to perform under theappropriate conditions the one or more chemical reactions thatchemically characterize the functional group.

Structural units forming polymers containing polyols comprise monomericpolyols such as pentaerythritol, ethylene glycol and glycerin. Exemplarypolymers containing polyols comprise polyesters, polyethers andpolysaccharides. Exemplary suitable polyethers include but are notlimited to diols and in particular diols with the general formulaHO—(CH₂CH₂O)_(p)—H with p≧1, such as polyethylene glycol, polypropyleneglycol, and poly(tetramethylene ether)glycol. Exemplary, suitablepolysaccharides include but are not limited to cyclodextrins, starch,glycogen, cellulose, chitin and β-Glucans. Exemplary, suitablepolyesters include but are not limited to polycarbonate, polybutyrateand polyethylene terephthalate, all terminated with hydroxyl end groups.Exemplary polymers containing polyols comprise polymers of about 500,000or less molecular weight and in particular from about 300 to about100,000.

Several polymers containing polyols are commercially available and/orcan be produced using techniques and procedures identifiable by askilled person. Exemplary procedures for the synthesis of an exemplarypolyol polymer are described in Liu et al 2005, and others areillustrated in Examples 1-4. Additional procedures for making polymercontaining polyols will be identifiable by a skilled person in view ofthe present disclosure.

The term “polymer containing a boronic acid” or “BA polymer” as usedherein indicates polymer containing at least one boronic acid grouppresented for binding to a hydroxyl group of a polymer containingpolyols. In particular, polymers containing boronic acids of thenanoparticles herein described include a polymer comprising in at leastone structural unit an alkyl or aryl substituted boronic acid containinga carbon to boron chemical bond. Suitable BA polymers comprise polymerswherein boronic acid is in a terminal structural unit or in any othersuitable position to provide the resulting polymer with hydrophilicproperties. Exemplary polymers containing polyols comprise polymers ofabout 40,000 or less molecular weight and in particular of about 20,000or less, or about 10,000 or less.

Several polymer containing a boronic acids are commercially availableand/or can be produced using techniques and procedures identifiable by askilled person. Exemplary procedures for the synthesis of an exemplarypolyol polymer are described in Liu and Reineke (2005) and other newones are illustrated in Examples 5-8. Additional procedures for makingBA polymers will be identifiable by a skilled person in view of thepresent disclosure.

In the nanoparticles herein described polyols polymers are coupled tothe BA polymers. The term “coupled” or “coupling” as used herein withreference to attachment between two molecules indicates an interactionforming a reversible covalent linkage. In particular, in presence of asuitable medium, a boronic acid presented on the BA polymer interactwith hydroxyl groups of the polyols via a rapid and reversible pair-wisecovalent interaction to form boronic esters in a suitable medium.Suitable medium include water and several aqueous solutions andadditional organic media identifiable by a skilled person. Inparticular, when contacted in an aqueous medium BA polymers and polyolspolymers react, producing water as a side product. The boronic acidpolyol interaction is generally more favorable in aqueous solutions butis also known to proceed in organic media. In addition, cyclic estersformed with 1,2 and 1,3 diols are generally more stable than theiracyclic ester counterparts.

Accordingly, in a nanoparticle herein described, at least one boronicacid of the polymer containing a boronic acid is bound to hydroxylgroups of the polymer containing a polyol with a reversible covalentlinkage. Formation of a boronic ester between BA polymers and polyolspolymers can be detected by methods and techniques identifiable by askilled person such as boron-11 nuclear magnetic resonance (¹¹B NMR),potentiomeric titration, UV/Vis and fluorescent detection techniqueswhereby the technique of choice is dependent on the specific chemicalnature and properties of the boronic acid and polyol composing thenanoparticle.

A nanoparticle resulting from coupling interactions of a BA polymerherein described with a polyol polymer herein described presents the BApolymer on the surface of the particle. In several embodiments thenanoparticles can have a diameter from about 1 to about 1000 nm and aspherical morphology although the dimensions and morphology of theparticle are largely determined by the specific BA polymer and polyolpolymers used to form the nanoparticles and by the compounds that arecarried on the nanoparticles according to the present disclosure.

In several embodiments the compound of interest carried by thenanoparticle forms part of the BA polymer and/or the polyol polymers.Examples of such embodiments are provided by nanoparticles wherein oneor more atoms of a polymer is replaced by a specific isotope e.g. ¹⁹Fand ¹⁰B, and are therefore suitable as agent for imaging the targetand/or providing radiation treatment to the target.

In several embodiments, the compound of interest carried thenanoparticle is attached to a polymer, typically a polyol polymer,through covalent or non covalent linkage. Examples of such embodimentsare provided by nanoparticles wherein one or more moieties in at leastone of the polyol polymer and BA polymer attaches one or more compoundsof interest.

The term “attach”, “attached” or “attachment” as used herein, refers toconnecting or uniting by a bond, link, force or tie in order to keep twoor more components together, which encompasses either direct or indirectattachment such that for example where a first compound is directlybound to a second compound, and the embodiments wherein one or moreintermediate compounds, and in particular molecules, are disposedbetween the first compound and the second compound.

In particular, in some embodiments a compound can be attached to thepolyol polymer or BA polymer through covalent linkage of the compound tosuitable moieties of the polymer. Exemplary covalent linkages areillustrated in Example 19 where, attachment of the drug Camptothecin toMucic Acid polymer is performed through biodegradable ester bondlinkage, and in Example 9, wherein attachment of transferrin toBA-PEG₅₀₀₀ is performed through pegylation of the transferrin.

In some embodiments, the polymer can be designed or modified to enablethe attachment of a specific compound of interest, for example by addingone or more functional groups able to specifically bind a correspondingfunctional group on the compound of interest. For example, in severalembodiments it is possible to PEGylate the nanoparticle with a BA-PEG-X,where X can be a Maleimide or an iodoacetyl group or any leaving groupthat will react specifically with a thiol or non-specifically with anamine. The compound to be attached can then react to the maleimide oriodoacetyl groups after modification to express a thiol functionalgroup. The compound to be attached can also be modified with aldehydesor ketone groups and these can react via a condensation reaction withthe diols on the polyols to give acetals or ketals.

In some embodiments, a compound of interest can be attached to thepolyol polymer or BA polymer through non covalent bonds such as ionicbonds and intermolecular interactions, between a compound to be attachedand a suitable moiety of the polymer. Exemplary non covalent linkagesare illustrated in Example 10.

A compound of interest can be attached to the nanoparticle before, uponor after formation of the nanoparticle, for example via modification ofa polymer and/or of any attached compound in the particulate composite.Exemplary procedures to perform attachment of a compound on thenanoparticle are illustrated in the Examples section. Additionalprocedures to attach a compound to a BA polymer polyol polymer or othercomponents of the nanoparticle herein described (e.g. a previouslyintroduced compound of interest) can be identified by a skilled personupon reading of the present disclosure.

In some embodiments, at least one compound of interest attached to a BApolymer presented on the nanoparticle herein described is an agent thatcan be used as a targeting ligand. In particular, in severalembodiments, the nanoparticle attaches on the BA polymer one or moreagents to be used as a targeting ligand, and on the polyol polymerand/or the BA polymer, one or more agents to be delivered to a target ofchoice.

The term “targeting ligand” as used in the present disclosure indicatesany molecule that can be presented on the surface of a nanoparticle forthe purpose of engaging a specific target, and in particular specificcellular recognition, for example by enabling cell receptor attachmentof the nanoparticle. Examples of suitable ligands include, but are notlimited to, vitamins (e.g. folic acid), proteins (e.g. transferrin, andmonoclonal antibodies), monosaccharides (e.g. galactose), peptides, andpolysaccharides. In particular targeting ligands can be antibodiesagainst certain surface cell receptors such as anti-VEGF, smallmolecules such as folic acid and other proteins such asholo-transferrin.

The choice of ligand, as one of ordinary skill appreciates, may varydepending upon the type of delivery desired. As another example, theligand may be membrane permeabilizing or membrane permeable agent suchas the TAT protein from HIV-1. The TAT protein is a viraltranscriptional activation that is actively imported into the cellnucleus. Torchilin, V. P. et al, PNAS. 98, 8786 8791, (2001). Suitabletargeting ligands attached to a BA polymer typically comprise a flexiblespacer such as a poly(ethylene oxide) with a boronic acid attached toits distal end (see Example 9).

In several embodiments, at least one of the compounds comprised orattached to the polyol polymer and/or BA polymer (including a targetingligand) can be an agent and in particular a drug, to be delivered to atarget, for example an individual, to which the chemical or biologicalactivity, e.g. the therapeutic activity, is to be exerted.

Selection of a polyol polymer and a BA polymer suitable to form ananoparticle herein described can be performed in view of the compoundand the target of interest. In particular, selection of a suitablepolymer containing a polyol and a suitable BA polymer to form ananoparticle herein described can be performed by providing candidatepolyol polymers and BA polymer, and selecting the polyol polymer and theBA polymer able to form a coupling interaction in the sense of thedisclosure, wherein the selected BA polymer and polyol polymer have achemical composition such that in view of the compound of interest andtargeting ligand to comprised or attached to the polyol polymers and/orthe BA polymers, the polyol polymers is less hydrophilic than the BApolymer. Detection of the BA polymer on the surface of the nanoparticleand related presentation on the environment external to the nanoparticlecan be performed by detection of the zeta potential which candemonstrate modification of the surface of the nanoparticle asillustrated in Example 12. (see in particular FIG. 6) Additionalprocedures to detect the surface charge of the particles and thestability of the particles in salt solutions, include detection ofchanges of the particle size such as the ones exemplified in Example 12(see in particular FIG. 7) and additional procedures identifiable by askilled person

In several embodiments, polymers containing polyols comprise one or moreof at least one of the following structural units

wherein

A is an organic moiety of formula

in which

-   -   R₁ and R₂ are independently selected from any carbon based or        organic group with a molecular weight of about 10 kDa or less;    -   X is independently selected from an aliphatic group, containing        one or more of —H, —F, —C, —N or —O; and    -   Y is independently selected from —OH or an organic moiety        bearing a hydroxyl (—OH) group including but not limited to        —CH₂OH, —CH₂CH₂OH, —CF₂OH, —CF₂CF₂OH, and C(R₁G₁)(RG₂)(R₁G₃)OH,        with R₁G₁, R₁ G₂ and R₁ G₃ are independently organic based        functionalities,        and

B is an organic moiety linking one of R₁ and R₂ of a first A moiety withone of the R₁ and R₂ of a second A moiety.

The term “moiety” as used herein indicates a group of atoms thatconstitute a portion of a larger molecule or molecular species. Inparticular, a moiety refers to a constituent of a repeated polymerstructural unit. Exemplary moieties include acid or base species,sugars, carbohydrates, alkyl groups, aryl groups and any other molecularconstituent useful in forming a polymer structural unit.

The term “organic moiety” as used herein indicates a moiety whichcontains a carbon atom. In particular, organic groups include naturaland synthetic compounds, and compounds including heteroatoms. Exemplarynatural organic moieties include but are not limited to most sugars,some alkaloids and terpenoids, carbohydrates, lipids and fatty acids,nucleic acids, proteins, peptides and amino acids, vitamins and fats andoils. Synthetic organic groups refer to compounds that are prepared byreaction with other compounds.

In several embodiments, one or more compounds of interest can beattached to (A), to (B) or to (A) and (B).

In several embodiments, R₁ and R₂ independently have the formula:

wherein

d is from 0 to 100

e is from 0 to 100

f is from 0 to 100,

Z is a covalent bond that links one organic moiety to another and inparticular to another moiety A or a moiety B as herein defined. and

Z₁ is independently selected from —NH₂, —OH, —SH, and —COOH

In several embodiments, Z can independently be selected from —NH—,—C(═O)NH—, —NH—C(═O), —SS—, —C(═O)O—, —NH(═NH₂ ⁺)— or —O—C(═O)—

In several embodiments where the structural unit A of a polymercontaining a polyol has formula (IV), X can be C_(v)H_(2v+1), wherev=0-5 and Y can be —OH

In some embodiments, R1 and/or R2 have formula (V) where Z is —NH(═NH₂⁺)— and/or Z₁ is NH₂.

In several embodiments, in polymers containing a polyol of the particleherein described (A) can be independently selected from the formulas

wherein

the spacer is independently selected from any organic moiety, and inparticular can include alkyl, phenyl or alkoxy groups optionallycontaining a heteroatom, such as sulfur, nitrogen, oxygen or fluorine;

the amino acid is selected from any organic group bearing a free amineand a free carboxylic acid group;

n is from 1 to 20; and

Z₁ is independently selected from —NH₂, —OH, —SH, and —COOH.

In several embodiments, Z1 is NH2, and/or the sugar can be anymonosaccharide such as glucose, fructose, mannitol, sucrose, galactose,sorbitol, xylose or galactose.

In several embodiments, in polymers containing a polyol of the particleherein described one ore more structural units (A) can independentlyhave the formula

In several embodiments, (B) can be formed by any straight, branched,symmetric or asymmetric compound linking the two (A) moieties throughfunctional groups.

In several embodiments, (B) can be formed by a compound where at leasttwo cross-linkable groups linking the two (A) moieties.

In some embodiments, (B) contains a neutral, cationic or anionic organicgroup whose nature and composition is dependent on the chemical natureof the compound to be covalently or non-covalently tethered

Exemplary cationic moieties of (B) for use with anionic cargo include,but are not limited to, organic groups bearing amidines groups,quartenary ammoniums, primary amine group, secondary amine group,tertiary amine groups (protonated below their pKa's), and immidazoliums

Exemplary anionic moieties contained in (B) for use with cationic cargoinclude, but are not limited to, organic groups bearing sulfonates offormula, nitrates of formula, carboxylates of formula, and phosphonates

In particular one or more cationic or anionic moieties (B) for use withanionic cargo and cationic cargos respectively can independently have ageneral formula of:

wherein R₅ is an electrophilic group that can be covalently linked to Awhen A contains nucleophilic groups. Examples of R₅ in this case includebut are not limited to—C(═O)OH, —C(═O)Cl, —C(═O)NHS, —C(═NH₂ ⁺)OMe, —S(═O)OCl—, —CH₂Br, alkyland aromatic esters, terminal alkynes, tosylate, and mesylate amongstseveral others. In the case where moiety A contains electrophilic endgroups, R₅ will bear nucleophilic groups such as —NH₂ (primary amines),—OH, —SH, N₃ and secondary amines.

In particular, when moiety (B) is a cationic moiety (B) for use withanionic cargo the “organic group” is an organic moiety that can have abackbone with a general formula consisting of C_(m)H_(2m) with m≧1 andother heteroatoms and must contain at least one of the followingfunctional groups including amidines of formula (XIII), quartenaryammoniums of formula (XIV), primary amine group of formula (XV),secondary amine group of formula (XVI), tertiary amine groups of formula(XVII) (protonated below their pKa's), and immidazoliums of formula(XVIII)

In embodiments, when moiety (B) is an anionic moiety (B) for use withcationic cargo, the “organic group” may have a backbone with a generalformula consisting of C_(m)H_(2m) with m≧1 and other heteroatoms andmust contain at least one of the following functional groups includingsulfonates of formula (XIX), nitrates of formula (XX), carboxylates offormula (XXI), and phosphonates of formula (XXII)

In embodiments wherein (B) is comprised by carboxylates (XXI), acompound containing primary amine or hydroxyl groups can also beattached via the formation of a peptide or an ester bond.

In embodiments wherein (B) is comprised of primary amine group offormula (XV), and/or secondary amine group of formula (XVI), a compoundcontaining carboxylic acid groups can also be attached via the formationof a peptide bond.

In several embodiments moiety (B) can independently be selected from

in which

q is from 1 to 20; and in particular can be 5

p is from 20 to 200; and

L is a leaving group.

The term “leaving group” as used herein indicates a molecular fragmentthat departs with a pair of electrons in heterolytic bond cleavage. Inparticular, a leaving group can be anions or neutral molecules, and theability of a leaving group to depart is correlated with the pK_(a) ofthe conjugate acid, with lower pK_(a) being associated with betterleaving group ability. Exemplary anionic leaving groups include halidessuch as Cl⁻, Br⁻, and I⁻, and sulfonate esters, such aspara-toluenesulfonate or “tosylate” (TsO⁻). Exemplary neutral moleculeleaving groups are water (H₂O), ammonia (NH₃), and alcohols (ROH).

In particular, in several embodiments, L can be a chloride (Cl), methoxy(OMe), t butoxy (OtBU) or N hydrosuccinimide (NHS).

In some embodiments the structural unit of formula (I) can have formula

In some embodiments the structural unit of formula (II) can have formula

In some embodiments the structural unit of formula (III) can haveformula

in which

n is from 1 to 20 and in particular from 1 to 4.

In some embodiments, the polymer containing polyol can have the formula

In some embodiments, the polymer containing a boronic acid contains atleast one terminal boronic acid group and has the following structure:

wherein

-   -   R₃ and R₄ can be independently selected from any hydrophilic        organic polymer, and in particular can independently be any        poly(ethylene oxides), and zwitterionic polymers.    -   X₁ can be an organic moiety containing one or more of —CH, —N,        or —B    -   Y₁ can be an alkyl group with a formula —C_(m)H_(2m)— with m≧1,        possibly containing olefins or alkynyl groups, or an aromatic        group such as a phenyl, biphenyl, napthyl or anthracenyl    -   r is from 1 to 1000,    -   a is from 0 to 3, and    -   b is from 0 to 3        and wherein functional group 1 and functional group 2 are the        same or different and are able to bind to a targeting ligand,        and in particular a protein, antibody or peptide, or is an end        group such as —OH, —OCH₃ or —(X₁)—(Y₁)—B(OH)₂—

In some embodiments, R₃ and R₄ are (CH₂CH₂O)_(t), where t is from 2 to2000 and in particular from 100 to 300

In some embodiments X₁ can be —NH—C(═O)—, —S—S—, —C(═O)—NH—, —O—C(═O)—or —C(═O)—O— and/or Y₁ can be a phenyl group.

In some embodiments r can be 1, a can be 0 and b can be 1.

In some embodiments, functional group 1 and functional group 2 are thesame or different and are independently selected from. —B(OH)₂, —OCH₃,—OH.

In particular, functional group 1 and/or 2 of formula (XXXI) can be afunctional group able to bind a cargo and in particular a targetingligand such as a protein, antibody or peptide, or can be an end groupsuch as —OH, —OCH₃ or —(X)—(Y)—B(OH)₂.

The term “functional group” as used herein indicates specific groups ofatoms within a molecular structure or portion thereof that areresponsible for the characteristic chemical reactions of that structureor portion thereof. Exemplary functional groups include hydrocarbons,groups containing halogen, groups containing oxygen, groups containingnitrogen and groups containing phosphorus and sulfur all identifiable bya skilled person. In particular, functional groups in the sense of thepresent disclosure include a carboxylic acid, amine, triarylphosphine,azide, acetylene, sulfonyl azide, thio acid and aldehyde. In particular,for example, a functional group able to bind a corresponding functionalgroup in a targeting ligand can be selected to comprise the followingbinding partners: carboxylic acid group and amine group, azide andacetylene groups, azide and triarylphosphine group, sulfonyl azide andthio acid, and aldehyde and primary amine. Additional functional groupscan be identified by a skilled person upon reading of the presentdisclosure. As used herein, the term “corresponding functional group”refers to a functional group that can react to another functional group.Thus, functional groups that can react with each other can be referredto as corresponding functional groups.

An end-group indicates a constitutional unit that is an extremity of amacromolecule or oligomer molecule. For example the end-group of a PETpolyester may be an alcohol group or a carboxylic acid group. End groupscan be used to determine molar mass. Exemplary end groups comprise —OH.—COOH, NH₂, and OCH₃,

In some embodiments, the polymer containing boronic acid can haveformula

wherein s is from 20 to 300.

Exemplary agents and targeting ligands that can be attached tonanoparticles of the present disclosure comprise organic or inorganicmolecules, including polynucleotides, nucleotides, aptamerspolypeptides, proteins, polysaccharides macromolecular complexesincluding but not limited to those comprising a mixture of protein andpolynucleotides, saccharides and/or polysaccharides, viruses, moleculeswith radioisotopes, antibodies or antibody fragments.

The term “polynucleotide” as used herein indicates an organic polymercomposed of two or more monomers including nucleotides, nucleosides oranalogs thereof. The term “nucleotide” refers to any of severalcompounds that consist of a ribose or deoxyribose sugar joined to apurine or pyrimidine base and to a phosphate group and that is the basicstructural unit of nucleic acids. The term “nucleoside” refers to acompound (such as guanosine or adenosine) that consists of a purine orpyrimidine base combined with deoxyribose or ribose and is foundespecially in nucleic acids. The term “nucleotide analog” or “nucleosideanalog” refers respectively to a nucleotide or nucleoside in which oneor more individual atoms have been replaced with a different atom or awith a different functional group. Accordingly, the term“polynucleotide” includes nucleic acids of any length, and in particularDNA, RNA, analogs and fragments thereof. A polynucleotide of three ormore nucleotides is also called “nucleotidic oligomer” or“oligonucleotide.”

The term “aptamers” as used here indicates oligonucleic acid or peptidemolecules that bind a specific target. In particular, nucleic acidaptamers can comprise, for example, nucleic acid species that have beenengineered through repeated rounds of in vitro selection orequivalently, SELEX (systematic evolution of ligands by exponentialenrichment) to bind to various molecular targets such as smallmolecules, proteins, nucleic acids, and even cells, tissues andorganisms. Aptamers are useful in biotechnological and therapeuticapplications as they offer molecular recognition properties that rivalthat of the antibodies. Peptide aptamers are peptides that are designedto specifically bind to and interfere with protein-protein interactionsinside cells. In particular, peptide aptamers can be derived, forexample, according to a selection strategy that is derived from theyeast two-hybrid (Y2H) system. In particular, according to thisstrategy, a variable peptide aptamer loop attached to a transcriptionfactor binding domain is screened against the target protein attached toa transcription factor activating domain. In vivo binding of the peptideaptamer to its target via this selection strategy is detected asexpression of a downstream yeast marker gene.

The term “polypeptide” as used herein indicates an organic linear,circular, or branched polymer composed of two or more amino acidmonomers and/or analogs thereof. The term “polypeptide” includes aminoacid polymers of any length including full length proteins and peptides,as well as analogs and fragments thereof. A polypeptide of three or moreamino acids is also called a protein oligomer, peptide or oligopeptide.In particular, the terms “peptide” and “oligopeptide” usually indicate apolypeptide with less than 50 amino acid monomers. As used herein theterm “amino acid”, “amino acidic monomer”, or “amino acid residue”refers to any of the twenty naturally occurring amino acids, non-naturalamino acids, and artificial amino acids and includes both D an L opticalisomers. In particular, non-natural amino acids include D-stereoisomersof naturally occurring amino acids (these including useful ligandbuilding blocks because they are not susceptible to enzymaticdegradation). The term “artificial amino acids” indicate molecules thatcan be readily coupled together using standard amino acid couplingchemistry, but with molecular structures that do not resemble thenaturally occurring amino acids. The term “amino acid analog” refers toan amino acid in which one or more individual atoms have been replaced,either with a different atom, isotope, or with a different functionalgroup but is otherwise identical to original amino acid from which theanalog is derived. All of these amino acids can be syntheticallyincorporated into a peptide or polypeptide using standard amino acidcoupling chemistries. The term “polypeptide” as used herein includespolymers comprising one or more monomer, or building blocks other thanan amino acid monomer. The terms monomer, subunit, or building blocksindicate chemical compounds that under appropriate conditions can becomechemically bonded to another monomer of the same or different chemicalnature to form a polymer. The term “polypeptide” is further intended tocomprise a polymer wherein one or more of the building blocks iscovalently bound to another by a chemical bond other than amide orpeptide bond.

The term “protein” as used herein indicates a polypeptide with aparticular secondary and tertiary structure that can participate in, butnot limited to, interactions with other biomolecules including otherproteins, DNA, RNA, lipids, metabolites, hormones, chemokines, and smallmolecules. Exemplary proteins herein described are antibodies.

The term “antibody” as used herein refers to a protein of the kind thatis produced by activated B cells after stimulation by an antigen and canbind specifically to the antigen promoting an immune response inbiological systems. Full antibodies typically consist of four subunitsincluding two heavy chains and two light chains. The term antibodyincludes natural and synthetic antibodies, including but not limited tomonoclonal antibodies, polyclonal antibodies or fragments thereof.Exemplary antibodies include IgA, IgD, IgG1, IgG2, IgG3, IgM and thelike. Exemplary fragments include Fab Fv, Fab′ F(ab′)2 and the like. Amonoclonal antibody is an antibody that specifically binds to and isthereby defined as complementary to a single particular spatial andpolar organization of another biomolecule which is termed an “epitope”.In some forms, monoclonal antibodies can also have the same structure. Apolyclonal antibody refers to a mixture of different monoclonalantibodies. In some forms, polyclonal antibodies can be a mixture ofmonoclonal antibodies where at least two of the monoclonal antibodiesbinding to a different antigenic epitope. The different antigenicepitopes can be on the same target, different targets, or a combination.Antibodies can be prepared by techniques that are well known in the art,such as immunization of a host and collection of sera (polyclonal) or bypreparing continuous hybridoma cell lines and collecting the secretedprotein (monoclonal).

In several embodiments, polyol polymers form a non-covalent complex orlinkage with one or more compounds of interest to be delivered accordingto the schematic illustration of FIGS. 1 and 2.

In several embodiments, a nanoparticle structure comprises an agent anda polymer containing a polyol, where the agent is linked to a polyolpolymer by a covalent bond. An example of a polyol polymer conjugated toan agent is detailed in Examples 16-21. In these embodiments, polyolpolymers conjugated to an agent (herein referred to as “polyolpolymer-agent conjugate”) form nanoparticles whose structure presentssites on their surface for interaction with BA molecules.

In several of those embodiments, the nanoparticle further comprises BApolymers configured to provide steric stabilization and/or targetingfunctionality to the nanoparticle. In particular, in those embodimentsthe addition of a BA polymer allows minimizing of self-aggregation andundesired interactions with other nanoparticles, thus providing enhancedsalt and serum stability. For example, steric stabilization is hereinprovided by the BA polymer having PEG as illustrated by the exemplarynanoparticle described in Example 12.

In such embodiments, the structure of this nanoparticle affords severaladvantages over agents delivery methods of the prior art, such as theability to provide controlled release of one or more agents. Thisfeature can be provided, for example, by the use of a biodegradableester linkage between the agent and the polyol polymer. A person skilledin the art will recognize other potential linkages suitable for thispurpose. In these embodiments, another advantage is the ability toprovide specific targeting of the agent through the BA polymer moiety.

In several embodiments, BA polymers may comprise a fluorinated boronicacid (Example 7) or a fluorinated cleavable boronic acid (Example 8)capable of being used as an imaging agent in MRI or other similartechniques. Such an imaging agent may be useful for tracking thepharmacokinetics or pharmacodynamics of an agent delivered by thenanoparticle.

In several embodiments, a nanoparticle structure comprises an agent anda polyol polymer, where the nanoparticle is a modified liposome. Inthese embodiments, the modified liposome comprises lipids conjugated topolyol polymers via a covalent linkage such that the surface of theliposome presents polyol polymers. In these embodiments, the modifiedliposomes form such that the agents to be delivered are contained withinthe liposome nanoparticle.

The term “liposome” as used herein indicates a vesicular structurecomprised of lipids. The lipids typically have a tail group comprising along hydrocarbon chain and a hydrophilic head group. The lipids arearranged to form a lipid bilayer with an inner aqueous environmentsuitable to contain an agent to be delivered. Such liposomes present anouter surface that may comprise suitable targeting ligands or moleculesfor specific recognition by cell surface receptors or other targets ofinterest.

The term “conjugated” as used herein indicates that one molecule hasformed a covalent bond with a second molecule and includes linkageswhere atoms covalently bond with alternating single and multiple (e.g.double) bonds (e.g., C═C—C═C—C) and influence each other to produceelectron delocalization.

In yet other embodiments of the present disclosure, a nanoparticlestructure comprises an agent and a polyol, where the nanoparticle is amodified micelle. In these embodiments, the modified micelle comprisespolyol polymers modified to contain a hydrophobic polymer block.

The term “hydrophobic polymer block” as used in the present disclosureindicates a segment of the polymer that on its own would be hydrophobic.

The term “micelle” as used herein refers to an aggregate of moleculesdispersed in a liquid. A typical micelle in aqueous solution forms anaggregate with the hydrophilic “head” regions in contact withsurrounding solvent, sequestering the hydrophobic single tail regions inthe micelle centre. In the present disclosure the head region may be,for example, a surface region of the polyol polymer while the tailregion may be, for example, the hydrophobic polymer block region of thepolyol polymer.

In these embodiments, polyol polymers with a hydrophobic polymer block,when mixed with an agent to be delivered, arrange to form a nanoparticlethat is a modified micelle with agents to be delivered contained withinthe nanoparticle. Such nanoparticle embodiments present polyol polymerson their surface that are suitable to interact with BA polymers that door do not have targeting functionality according to previousembodiments. In these embodiments, BA polymers capable of use for thispurpose include those with hydrophilic A and hydrophobic B in formula(I) or (II). This interaction provides the same or similar advantages asit does for other nanoparticle embodiments mentioned above.

In some embodiments, nanoparticles or related components can becomprised in a composition together with an acceptable vehicle. The term“vehicle” as used herein indicates any of various media acting usuallyas solvents, carriers, binders, excipients or diluents for ananoparticle comprised in the composition as an active ingredient.

In some embodiments, where the composition is to be administered to anindividual the composition can be a pharmaceutical composition and theacceptable vehicle can be a pharmaceutically acceptable vehicle.

In some embodiments, a nanoparticle can be included in pharmaceuticalcompositions together with an excipient or diluent. In particular, insome embodiments, pharmaceutical compositions are disclosed whichcontain nanoparticle, in combination with one or more compatible andpharmaceutically acceptable vehicle, and in particular withpharmaceutically acceptable diluents or excipients.

The term “excipient” as used herein indicates an inactive substance usedas a carrier for the active ingredients of a medication. Suitableexcipients for the pharmaceutical compositions herein disclosed includeany substance that enhances the ability of the body of an individual toabsorb the nanoparticle. Suitable excipients also include any substancethat can be used to bulk up formulations with nanoparticles to allow forconvenient and accurate dosage. In addition to their use in thesingle-dosage quantity, excipients can be used in the manufacturingprocess to aid in the handling of nanoparticles. Depending on the routeof administration, and form of medication, different excipients may beused. Exemplary excipients include but are not limited to antiadherents,binders, coatings disintegrants, fillers, flavors (such as sweeteners)and colors, glidants, lubricants, preservatives, sorbents.

The term “diluent” as used herein indicates a diluting agent which isissued to dilute or carry an active ingredient of a composition.Suitable diluents include any substance that can decrease the viscosityof a medicinal preparation.

In certain embodiments, compositions and, in particular, pharmaceuticalcompositions can be formulated for systemic administration, whichincludes parenteral administration and more particularly intravenous,intradermic, and intramuscular administration.

Exemplary compositions for parenteral administration include but are notlimited to sterile aqueous solutions, injectable solutions orsuspensions including nanoparticles. In some embodiments, a compositionfor parenteral administration can be prepared at the time of use bydissolving a powdered composition, previously prepared in a freeze-driedlyophilized form, in a biologically compatible aqueous liquid (distilledwater, physiological solution or other aqueous solution).

The term “lyophilization” (also known as freeze-drying orcryodesiccation) indicates a dehydration process typically used topreserve a perishable material or make the material more convenient fortransport. Freeze-drying works by freezing the material and thenreducing the surrounding pressure and adding enough heat to allow thefrozen water in the material to sublime directly from the solid phase togas.

In pharmaceutical applications freeze-drying is often used to increasethe shelf life of products, such as vaccines and other injectables. Byremoving the water from the material and sealing the material in a vial,the material can be easily stored, shipped, and later reconstituted toits original form for injection.

In several embodiments nanoparticles herein described are delivered to apredetermined target. In some embodiments, the target is an in vitrobiological system and the method comprises contacting target with thenanoparticle herein described.

In some embodiments, a method is provided for delivery of an agent to anindividual where the method comprises formulating a suitablenanoparticle according to various disclosed embodiments. Thenanoparticles may also be formulated into a pharmaceutically acceptablecomposition according to several disclosed embodiments. The methodfurther comprises delivering a nanoparticle to a subject. To deliver thenanoparticle to an individual, the nanoparticle or nanoparticleformulations may be given orally, parenterally, topically, or rectally.They are delivered in forms suitable for each administration route. Forexample, nanoparticle compositions can be administered in tablets orcapsule form, by injection, inhalation, eye lotion, ointment,suppository, infusion; topically by lotion or ointment; and rectally bysuppositories.

The term “individual” as used herein includes a single biologicalorganism including but not limited to plants or animals and inparticular higher animals and in particular vertebrates such as mammalsand in particular human beings.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradennal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrastemal, injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a nanoparticle or composition thereofother than directly into the central nervous system, such that it entersthe individual's system and, thus, is subject to metabolism and otherlike processes, for example, subcutaneous administration.

Actual dosage levels of the active ingredient or agent in thepharmaceutical compositions herein described may be varied so as toobtain an amount of the active ingredient which is effective to achievethe desired therapeutic response for a particular individual,composition, and mode of administration, without being toxic to theindividual.

These therapeutic polymer conjugate may be administered to humans andother animals for therapy by any suitable route of administration,including orally, nasally, as by, for example, a spray, rectally,intravaginally, parenterally, intracisternally and topically, as bypowders, ointments or drops, including buccally and sublingually.

Regardless of the route of administration selected, the therapeuticpolymer conjugate, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

In particular in some embodiments, the compound delivered is a drug fortreating or preventing a condition in the individual.

The term “drug” or “therapeutic agent” indicates an active agent thatcan be used in the treatment, prevention, or diagnosis of a condition inthe individual or used to otherwise enhance the individual's physical ormental well-being.

The term “condition” as used herein indicates a usually the physicalstatus of the body of an individual, as a whole or of one or more of itsparts, that does not conform to a physical status of the individual, asa whole or of one or more of its parts, that is associated with a stateof complete physical, mental and possibly social well-being. Conditionsherein described include but are not limited disorders and diseaseswherein the term “disorder” indicates a condition of the livingindividual that is associated to a functional abnormality of the body orof any of its parts, and the term “disease” indicates a condition of theliving individual that impairs normal functioning of the body or of anyof its parts and is typically manifested by distinguishing signs andsymptoms. Exemplary conditions include but are not limited to injuries,disabilities, disorders (including mental and physical disorders),syndromes, infections, deviant behaviors of the individual and atypicalvariations of structure and functions of the body of an individual orparts thereof.

The term “treatment” as used herein indicates any activity that is partof a medical care for or deals with a condition medically or surgically.

The term “prevention” as used herein indicates any activity, whichreduces the burden of mortality or morbidity from a condition in anindividual. This takes place at primary, secondary and tertiaryprevention levels, wherein: a) primary prevention avoids the developmentof a disease; b) secondary prevention activities are aimed at earlydisease treatment, thereby increasing opportunities for interventions toprevent progression of the disease and emergence of symptoms; and c)tertiary prevention reduces the negative impact of an alreadyestablished disease by restoring function and reducing disease-relatedcomplications.

Exemplary compounds that can be delivered by the nanoparticles hereindescribed and that are suitable as drugs comprise compounds able to emitelectromagnetic radiations (such as ¹⁰B isotopes) to be used inradiation treatments (such as boron neutron capture) Additionaltherapeutic agents comprise any lipophilic or hydrophilic, synthetic ornaturally occurring biologically active therapeutic agent includingthose known in the art. The Merck Index, An Encyclopedia of Chemicals,Drugs, and Biologicals, 13th Edition, 2001, Merck and Co., Inc.,Whitehouse Station, N.J. Examples of such therapeutic agents include,but are not limited to, small molecule pharmaceuticals, antibiotics,steroids, polynucleotides (e.g. genomic DNA, cDNA, mRNA, siRNA, shRNA,miRNA, antisense oligonucleotides, viruses, and chimericpolynucleotides), plasmids, peptides, peptide fragments, small molecules(e.g. doxorubicin), chelating agents (e.g. deferoxamine (DESFERAL),ethylenediaminetetraacetic acid (EDTA)), natural products (e.g. Taxol,Amphotericin), and other biologically active macromolecules such as, forexample, proteins and enzymes. See also U.S. Pat. No. 6,048,736 whichlists active agents (therapeutic agents) that can be used as therapeuticagent with nanoparticles herein described. Small molecule therapeuticagents may not only be the therapeutic agent within the compositeparticle but, in an additional embodiment, may be covalently bound to apolymer in the composite. In several embodiments, the covalent bond isreversible (e.g. through a prodrug form or biodegradable linkage such asa disulfide) and provides another way of delivering the therapeuticagent. In several embodiments therapeutic agent that can be deliveredwith the nanoparticles herein described include chemotherapeutics suchas epothilones, camptothecin-based drugs, taxol, or a nucleic acid suchas a plasmid, siRNA, shRNA, miRNA, antisense oligonucleotides aptamersor their combination, and additional drugs identifiable by a skilledperson upon reading of the present disclosure.

In some embodiments, the compound delivered is a compound suitable forimaging a cell or tissue of the individual. Exemplary compounds that canbe delivered by the nanoparticles herein described and that are suitablefor imaging comprise compounds that contain isotopes such as ¹⁹Fisotopes for MR imaging, ¹⁸F or ⁶⁴Cu for PET imaging etc.

In particular, the nanoparticles described herein can be configured tocontain ¹⁹F-containing BA polymers. For example, ¹⁹F atoms can beincorporated into a non-cleavable or cleavable BA polymer compound.Other locations for the ¹⁹F atoms are possible on the BA polymercomponent, the polyol polymer component, or on the agent to bedelivered. These and other variations will be apparent to one skilled inthe art.

In several embodiments, the nanoparticles herein described can be usedto deliver chemicals used in the agricultural industry. In anotherembodiment of the invention, the agent delivered by the nanoparticleherein described is a biologically active compound having microbiocidaland agricultural utility. These biologically active compounds includethose known in the art. For example, suitable agriculturallybiologically active compounds include, but are not limited to,fertilizers, fungicides, herbicides, insecticides, and mildewcides.Microbicides are also used in water-treatment to treat municipal watersupplies and industrial water systems such as cooling waters, whitewater systems in papermaking. Aqueous systems susceptible tomicrobiological attack or degradation are also found in the leatherindustry, the textile industry, and the coating or paint industry.Examples of such microbicides and their uses are described, individuallyand in combinations, in U.S. Pat. Nos. 5,693,631, 6,034,081, and6,060,466, which are incorporated herein by reference. Compositionscontaining active agents such as those discussed above may be used inthe same manner as known for the active ingredient itself. Notably,because such uses are not pharmacological uses, the polymer of thecomposite does not necessarily have to meet the toxicity profilerequired in pharmaceutical uses.

In certain embodiments, nanoparticles comprising polyol polymers and BApolymers can also be comprised in a system suitable for delivering anyof the compounds herein indicated and in particular agents, using ananoparticle. In some embodiments of the system, nanoparticles areprovided with components suitable for preparing the nanoparticles foradministration to an individual.

The systems herein disclosed can be provided in the form of kits ofparts. For example the polyol polymers and/or BA polymers can beincluded as a molecule alone or in the presence of suitable excipients,vehicles or diluents.

In a kit of parts, polyol polymers, BA polymers, and/or agents to bedelivered are comprised in the kit independently possibly included in acomposition together with suitable vehicle carrier or auxiliary agents.For example, polyol polymers and/or BA polymers can be included in oneor more compositions alone and/or included in a suitable vector. Also,an agent to be delivered can be included in a composition together witha suitable vehicle carrier or auxiliary agent. Alternatively, the agentmay be supplied by the end user and may be absent from the kit of parts.Furthermore, the polyol polymers, BA polymers and/or agents can beincluded in various forms suitable for appropriate incorporation into ananoparticle.

Additional components can also be included and comprise microfluidicchip, reference standards, buffers, and additional componentsidentifiable by a skilled person upon reading of the present disclosure.

In the kit of parts herein disclosed, the components of the kit can beprovided, with suitable instructions and other necessary reagents, inorder to perform the methods here disclosed. In some embodiments, thekit can contain the compositions in separate containers. Instructions,for example written or audio instructions, on paper or electronicsupport such as tapes or CD-ROMs, for carrying out the assay, can alsobe included in the kit. The kit can also contain, depending on theparticular method used, other packaged reagents and materials (such aswash buffers and the like).

Further details concerning the identification of the suitable carrieragent or auxiliary agent of the compositions, and generallymanufacturing and packaging of the kit, can be identified by the personskilled in the art upon reading of the present disclosure.

In some embodiments, a nanoparticle may be prepared by preparing theindividual components of the nanoparticle followed by mixing thecomponents in various orders to arrive at a desired compositenanoparticle structure. Preparation and mixing of components is carriedout in suitable solutions known by those skilled in the art.

The term “mixing” as used herein indicates addition of one solutioncomprising a molecule of interest with another solution comprisinganother molecule of interest. For example, an aqueous solution of polyolpolymers may be mixed with an aqueous solution of BA polymers in thecontext of the present disclosure.

The term “solution” as used herein indicates any liquid phase samplecontaining molecules of interest. For example, an aqueous solution ofpolyol polymers may comprise polyol polymers diluted in water or anybuffered solution, in particular aqueous solutions.

In some embodiments, a nanoparticle can be prepared by mixing polyolpolymers with an agent to be delivered (FIGS. 1 and 2), forming a polyolpolymer-agent nanoparticle. In other embodiments, a nanoparticle may beprepared by further mixing BA polymers with the polyol polymer-agentnanoparticle. In other embodiments, a nanoparticle is prepared by mixingpolyol polymers with BA polymers, followed by mixing an agent to bedelivered. In yet other embodiments, a nanoparticle is prepared bysimultaneously mixing polyol polymers, BA polymers, and an agent to bedelivered.

In some embodiments, a nanoparticle is prepared by forming a polyolpolymer-agent conjugate according to various embodiments of the presentdisclosure, thus preparing a nanoparticle comprised of a polyolpolymer-agent conjugate. In other embodiments nanoparticles comprised ofa polyol polymer-agent conjugates may be prepared by dissolving thenanoparticles in a suitable aqueous solution. In yet furtherembodiments, nanoparticles comprised of a polyol polymer-agentconjugates may be prepared by mixing polyol polymer-agent conjugateswith BA polymers that do or do not provide targeting ligand.

In some embodiments, a nanoparticle can be prepared by mixing polyolpolymers with a hydrophobic polymer block with an agent to be delivered,thus preparing a modified micelle according to embodiments of thepresent disclosure. In other embodiments, a nanoparticle may be preparedby further mixing the modified micelle with BA polymers. In yet otherembodiments, a nanoparticle may be prepared by mixing polyol polymerswith BA polymers, followed by mixing an agent to be delivered, thuspreparing a nanoparticle that is a modified micelle.

In some embodiments of the present disclosure, a nanoparticle can beprepared by mixing lipids conjugated with polyol polymers with BApolymers and/or agents to be delivered, thus preparing a modifiedliposome. In various embodiments, a nanoparticle may be prepared bymixing lipids conjugated with polyol polymers with BA polymers, followedby mixing agents to be delivered. In other embodiments, a nanoparticlemay be prepared by mixing lipids conjugated with polyol polymers withagents to be delivered. In other embodiments, a nanoparticle may beprepared by mixing lipids conjugated with polyol polymers with agents tobe delivered, followed by mixing BA polymers, thus preparing ananoparticle that is a modified liposome.

The formation of nanoparticles according to several embodiments of thepresent disclosure can be analyzed with techniques and procedures knownby those with skill in the art. For example, in several embodiments, gelretardation assays are used to monitor and measure the incorporation ofa nucleic acid agent within a nanoparticle (Example 10). In severalembodiments, a suitable nanoparticle size and/or zeta potential can bechosen using known methods (Example 11).

Further details concerning the identification of the suitable carrieragent or auxiliary agent of the compositions, and generallymanufacturing and packaging of the kit, can be identified by the personskilled in the art upon reading of the present disclosure.

EXAMPLES

The methods system herein described are further illustrated in thefollowing examples, which are provided by way of illustration and arenot intended to be limiting. A person skilled in the art will appreciatethe applicability of the features described in detail for methods ofnucleic acid detection and detection of other targets, such as proteins,antigens, eukaryotic or prokaryotic cells, and the like.

All chemical reagents were obtained from commercial suppliers and wereused as received without further purification. Polymer samples wereanalyzed on a Viscotek GPC System equipped with a TDA 302 tripledetector array consisting of a differential refractive index (RI)detector, a differential viscometer and a low angle light scatteringdetector. A 7.5% acetic acid solution was used as eluant at a 1 mL/minflow rate.

pGL3, a plasmid containing the firefly luciferase gene was extracted andpurified from bacteria expressing pGL3. siGL3 was purchased fromIntegrated DNA Technologies (sequence provided below). siCON1 (sequenceprovided below) was purchased from Dharmacon. HeLa cells were used todetermine the efficacy of pDNA or siRNA delivery by the cationic mucicacid diamine-DMS polymer.

TABLE 1 siRNA sequences Plasmid Sequences SEQ ID NO siGL3GUGCCAGAGUCCUUCGAUAdTdT SEQ ID NO: 1 (sense) UAUCGAAGGACUCUGGCACdTdTSEQ ID NO: 2 (antisense) siCON1 UAGCGACUAAACACAUCAAUU SEQ ID NO: 3(sense) UUGAUGUGUUUAGUCGCUAUU SEQ ID NO: 4 (antisense)

Example 1 Synthesis of Mucic Acid Dimethyl Ester, (1)

5 g (22.8 mmol) of mucic acid (Aldrich) was added to a 500 mL roundbottom flask containing 120 mL of methanol and 0.4 mL of concentratedsulfuric acid. This mixture was allowed to reflux at 85° C. overnightunder constant stirring. The mixture was subsequently filtered, washedwith methanol and then recrystallized from a mixture of 80 mL methanoland 0.5 mL triethylamine. After drying under vacuum overnight, 8.0 g(33.6 mmol, 71%) of mucic acid dimethyl ester was obtained. ¹H NMR((CD₃)₂SO) δ 4.88-4.91 (d, 2H), 4.78-4.81 (m, 2H), 4.28-4.31 (d, 2H),3.77-3.78 (d, 2H), 3.63 (s, 6H). ESI/MS (m/z): 261.0 [M+Na]⁺

Example 2 Synthesis of N—BOC-Protected Mucic Acid Diamine, (2)

A mixture of 8 g (33.6 mmol) of Mucic Acid Dimethyl Ester (1; Example1), 12.4 mL (88.6 mmol) triethylamine and 160 mL methanol was heatedunder reflux at 85° C. in a 500 mL round bottom flask under constantstirring for 0.5 h prior to the addition of 14.2 g (88.6 mmol) N—BOCdiamine (Fluka) dissolved in methanol (32 mL). This reaction suspensionwas then returned to reflux. After refluxing overnight, the mixture wasfiltered, washed with methanol, recrystallized from methanol and thendried under vacuum to yield 9.4 g (19 mmol, 57%) of N-BOC-ProtectedMucic Acid Diamine. ¹H NMR ((CD₃)₂SO) δ 7.66 (m, 2H), 6.79 (m, 2H),5.13-5.15 (d, 2H), 4.35-4.38 (d, 2H), 4.08-4.11 (m, 2H), 3.78-3.80 (d,2H), 2.95-3.15 (m, 8H), 1.38 (s, 18). ESI/MS (m/z): 517.1 [M+Na]⁺

Example 3 Synthesis of Mucic Acid Diamine, (3)

8 g (16.2 mmol) of the N—BOC-Protected Mucic Acid Diamine (2; Example 2)was transferred to a 500 mL round bottom flask containing 3 M HCl inmethanol (160 mL) and allowed to reflux overnight at 85° C. underconstant stirring. The precipitate was subsequently filtered, washedwith methanol and vacuum dried overnight to give 5.7 g (15.6 mmol, 96%)of Mucic Acid Diamine. ¹H NMR ((CD₃)₂SO) δ 7.97 (m, 8H), 5.35-5.38 (m,2H), 4.18-4.20 (m, 2H), 3.82 (m, 2H), 3.35-3.42 (m, 8H), 2.82-2.90 (m,4H). ESI/MS (m/z): 294.3 [M]⁺, 317.1 [M+Na]⁺, 333.0 [M+K]⁺

Example 4 Mucic Acid Diamine-DMS Copolymer (MAP), (4)

A 1.5 mL eppendorff tube was charged with a solution of 85.5 mg (0.233mmol) of the bis(hydrochloride) salt of Example 3 (3) in 0.8 mL of 0.1 MNaHCO₃. Dimethylsuberimidate.2HCl (DMS, Pierce Chemical Co., 63.6 mg,0.233 mmol) was added and the solution was vortexed and centrifuged todissolve the components. The resulting mixture was stirred at roomtemperature for 15 h. The mixture was then diluted to 8 mL with waterand the pH was brought to 4 with the addition of 1 N HCl. This solutionwas then dialyzed with a 3500 MWCO dialysis membrane (Pierce pleateddialysis tubing) in ddH₂O for 24 h. The dialyzed solution waslyophilized to dryness to give 49 mg of a white fluffy powder. ¹H NMR(500 MHz, dDMSO) δ 9.15 (bs), 7.92 (bs), 5.43 (bs), 4.58 (bs), 4.17(bs), 3.82 (bs), 3.37 (bs), 3.28 (bs), 2.82 (bs), 2.41 (bs), 1.61 (bs),1.28 (bs). ¹³C NMR (126 MHz, dDMSO) δ 174.88 (s, 1H), 168.38 (s, 1H),71.45 (s, 4H), 71.22 (s, 3H), 42.34 (s, 2H), 36.96 (s, 3H), 32.74 (s,3H), 28.09 (s, 4H), 26.90 (s, 4H). Mw [GPC]=2520, Mw/Mn=1.15.

Polymer 4 is an example of a cationic A-B type (repeating structure isABABAB . . . ) polymer containing a polyol.

Example 5 Boronic Acid-Amide-PEG₅₀₀₀, (5)

When a polymer of Example 4 is assembled with nucleic acids, e.g.,siRNAs, they will form nanoparticles. These nanoparticles will need tohave steric stabilization to be used in mammals and optionally theycould have targeting agents included. To perform these two functions,the nanoparticles can be decorated with PEG for steric stabilization andPEG-targeting ligands. To do so, PEG compounds containing boronic acidsare prepared. For example, a PEG containing boronic acid can besynthesized according the example below.

332 mg of 4-carboxyphenylboronic acid (2 mmol) was dissolved in 8 mL ofSOCl₂. To this was added a few drops of DMF and the mixture was refluxedunder argon for 2 h. Excess SOCl₂ was removed under reduced pressure andthe resulting solid was dissolved in 10 mL of anhydrous dichloromethane.To this solution was added 500 mg of PEG₅₀₀₀-NH₂ (2 mmol) and 418 μL oftriethylamine (60 mmol) dissolved in 5 mL of dichloromethane at 0° C.under argon. The resulting mixture was warmed to room temperature andstirring was continued overnight. The dichloromethane solvent wasremoved under reduced pressure and the resulting liquid was precipitatedwith 20 mL of diethyl ether. The precipitate was filtered, dried andre-dissolved in ddH₂O. The aqueous solution was then filtered with a0.45 μm filter and dialyzed with a 3500 MWCO dialysis membrane (Piercepleated dialysis tubing) in ddH₂O for 24 h. The dialyzed solution waslyophilized to dryness. ¹H NMR (300 MHz, dDMSO) δ 7.92-7.77 (m), 4.44(d), 4.37 (t), 3.49 (m), 2.97 (s).

Example 6 Boronic Acid-Disulfide-PEG₅₀₀₀, (6)

A cleavable version (under reducing conditions) of the PEG compound ofExample 5 can also be synthesized as follows.

250 mg of PEG₅₀₀₀-SH (0.05 mmol, LaySanBio Inc.) was added to a glassvial equipped with a stirbar. To this was added 110 mg of aldrithiol-2(0.5 mmol, Aldrich) dissolved in 4 mL of methanol. The solution wasstirred at room temperature for 2 h after which, 77 mg ofmercaptophenylboronic acid (0.5 mmol, Aldrich) in 1 mL of methanol wasadded. The resulting solution was stirred for an additional 2 h at roomtemperature. Methanol was removed under vacuuo and the residue wasre-dissolved in 2 mL of dichloromethane. 18 mL of diethyl ether wasadded to the dichloromethane solution and the mixture was allowed to sitfor 1 h. The resulting precipitate was collected via centrifugation,washed several times with diethyl ether and dried. The dried solid wasre-dissolved in water, filtered with a 0.45 μm filter and dialyzed witha 3500 MWCO dialysis membrane (Pierce pleated dialysis tubing) in ddH₂Ofor 15 h. The dialyzed solution was lyophilized to dryness. ¹H NMR (300MHz, dDMSO) δ 8.12-8.00 (m), 7.83-7.72 (m), 7.72-7.61 (m), 7.61-7.43(m), 3.72 (d, J=5.4), 3.68-3.15 (m), 3.01-2.83 (m).

Example 7 Synthesis of (2,3,5,6)-Tetrafluorophenyl Boronic Acid-PEG₅₀₀₀,(7)

A fluorinated version of the PEG compound containing boronic acids ofExample 5 can be synthesized and used as an imaging agent with thetherapeutic nanoparticle. The fluorine atoms for imaging can beincorporated as described and illustrated below.

(2,3,5,6)-fluorocarboxyphenylboronic acid is dissolved in excess SOCl₂(˜100 eq.) and to it is added a few drops of DMF. The mixture isrefluxed under argon for 2 h. Excess SOCl₂ is removed under reducedpressure and the resulting residue is dissolved in anhydrousdichloromethane. To this solution, PEG₅₀₀₀-NH₂ (1 eq.) and triethylamine(30 eq,) dissolved in dichloromethane is added at 0° C. under argon. Theresulting mixture is warmed to room temperature and stirring iscontinued overnight. The dichloromethane solvent is removed underreduced pressure and the resulting liquid is precipitated with diethylether. The precipitate is filtered, dried and re-dissolved in ddH₂O. Theaqueous solution is then filtered with a 0.45 μm filter and dialyzedwith a 3500 MWCO dialysis membrane (Pierce pleated dialysis tubing) inddH₂O for 24 h. The dialyzed solution is lyophilized to dryness.

The fluorine containing compound is useful to provide ¹⁹F in thenanoparticle. The ¹⁹F can be detected by magnetic resonance spectroscopyusing a standard patient MRI. The addition of the ¹⁹F enables thenanoparticles to be imaged (can be done for just imaging or with theaddition of a therapeutic agent can allow for imaging and therapy).

Example 8 Synthesis of (2,3,5,6)-Tetrafluorophenyl BoronicAcid-Disulfide-PEG₅₀₀₀, (8)

A fluorinated version of the cleavable PEG compound containing boronicacids of Example 5 can be synthesized and used as an imaging agent withthe therapeutic nanoparticle. The fluorine atoms for imaging can beincorporated as described and illustrated below.

250 mg of PEG₅₀₀₀-SH (0.05 mmol, LaySanBio Inc.) are added to a glassvial equipped with a stirbar. To this is added 110 mg of aldrithiol-2(0.5 mmol, Aldrich) dissolved in 4 mL of methanol. The solution isstirred at room temperature for 2 h after which, 77 mg of(2,3,5,6)-fluoro-4-mercaptophenylboronic acid (0.5 mmol) in 1 mL ofmethanol is added. The resulting solution is stirred for an additional 2h at room temperature. Methanol is removed under vacuuo and the residueis re-dissolved in 2 mL of dichloromethane. 18 mL of diethyl ether isadded to the dichloromethane solution and the mixture is allowed to sitfor 1 h. The resulting precipitate is collected via centrifugation,washed several times with diethyl ether and dried. The dried solid isre-dissolved in water, filtered with a 0.45 μm filter and dialyzed witha 3500 MWCO dialysis membrane (Pierce pleated dialysis tubing) in ddH₂Ofor 15 h. The dialyzed solution is lyophilized to dryness.

Example 9 Synthesis of Boronic Acid-PEG₅₀₀₀-Transferrin, (9)

A targeting agent could be placed at the other end of the PEG from theboronic acid in the compounds of Examples 5-8, for example according toan approach schematically illustrated in FIG. 12 with reference toattachment of transferrin.

Thus, the components of a system containing nucleic acids as thetherapeutic could be (targeting ligand could be a protein liketransferrin (FIG. 12), an antibody or antibody fragment, a peptide likeRGD or LHRH, a small molecule like folate or galactose, etc.). A boronicacid PEGylated targeting agent can be synthesized as follows.

In particular, to synthesize the Boronic Acid PEG₅₀₀₀-Transferrinaccording to the approach schematically illustrated in FIG. 12 thefollowing procedure was performed. A solution of 10 mg (0.13 μmol) ofHuman holo-Transferrin (iron rich) (Sigma Aldrich) in 1 mL of 0.1M PBSbuffer (p.H. 7.2) was added to 3.2 mg of OPSS-PEG₅₀₀₀-SVA (5 eq, 0.64μmol, LaysanBio Inc.). The resulting solution was stirred at roomtemperature for 2 h. The PEGylated Transferrin was purified from theunreacted OPSS-PEG₅₀₀₀-SVA using an Ultracel 50,000 MWCO (AmiconUltra-4, Millipore) and from unreacted Transferrin using a gelfiltration column G3000SWx1 (Tosoh Biosep) (confirmed by HPLC andMALDI-TOF analysis). 100 μg of the OPSS-PEG₅₀₀₀ PEGylated Transferrin in100 μL was then incubated at room temperature with 20 μL,4-mercaptophenylboronic acid (1 μg/μL, 20 μg, 100 eq.) for 1 h. Afterincubation, the solution was dialyzed twice with a YM-30,000 NMWI device(Millipore) to remove excess 4-mercaptophenylboronic and thepyridyl-2-thione by-product.

Example 10 Formulation of MAP-Nucleic Acid Particles—Gel RetardationAssay

As diagramed in FIG. 1, 1 μg of plasmid DNA or siRNA in DNAse and RNASefree water (0.1 μg/μL, 10 μL) was mixed with 10 μL of MAP at variousconcentrations in DNAse and RNASe free water to give charge ratios (“+”charge on polymer to “−” charge on nucleic acid) of 0.5, 1, 1.5, 2, 2.5,and 5. The resulting mixtures were incubated for 30 minutes at roomtemperature. 10 μL of the 20 μL solutions were loaded onto a 1% agarosegel with 3.5 μL of loading buffer and the gel was electrophoresed at 80V for 45 minutes as shown in FIGS. 3 and 4. Nucleic acid that is notcontained within the nanoparticle will migrate on the gel. These resultsgive guidance to the charge ratios necessary for nucleic acidcontainment within the nanoparticles.

Example 11 Particle Size and Zeta Potential of MAP-Nucleic AcidParticles

1 μg of plasmid DNA in DNAse and RNASe free water (0.1 μg/μL, 10 μL) wasmixed with 10 μL of MAP at various concentrations in DNAse and RNASefree water to give charge ratios of 0.5, 1, 1.5, 2, 2.5, and 5. Theresulting mixtures were incubated for 30 minutes at room temperature.The 20 μL mixture was then diluted with DNAse and RNASe free water to 70μL for particle size measurements. This 70 μL solution was then dilutedto 1400 μL with 1 mM KCl for zeta potential measurements. The particlesize and zeta potential measurements were made on a ZetaPals dynamiclight scattering (DLS) instrument (Brookhaven Instruments). The resultsare shown in FIG. 5.

Example 12 Particle Size Stabilization by PEGylation with Boronic AcidPEG_(5k)

As diagrammed in FIG. 2, 2 μg of plasmid DNA in DNAse and RNASe freewater (0.45 μg/μL, 4.4 μL) was diluted to 80 μL in DNAse and RNASe freewater. This plasmid solution was mixed with 4.89 μg of MAP (0.5 μg/μL,9.8 μL) also diluted to 80 μL in DNAse and RNASe free water to give a3+/− charge ratio and a final plasmid concentration of 0.0125 μg/μL. Theresulting mixture was incubated for 30 minutes at room temperature. Tothis solution was added 480 μg of boronic acid PEG_(5K), (compound 6;Example 6), (20 μg/μL, 24 μL). This mixture was then incubated furtherfor 30 minutes, dialyzed twice in DNAse and RNASe free water with a 0.5mL 100,000 MWCO membrane (BIOMAX, Millipore Corporation) andreconstituted in 160 μL of DNAse and RNASe free water. Half of thesolution was diluted with 1.4 mL of 1 mM KCl for zeta potentialmeasurements (FIG. 6). Note that the zeta potential of the BA containingnanoparticles show a lower zeta potential than the nanoparticles that donot. These results support the conclusion that the BA containingnanoparticles have the BA localized on the exterior of thenanoparticles. The other half was used to measure the particle size. Theparticle size was measured every minute for 5 minutes after which, 10.2μL of 10×PBS was added such that the final 90.2 μL solution was in1×PBS. The particle size was then measured again every minute foranother 10 minutes as shown in FIG. 7. The BA containing nanoparticlesseparated from non-particle components (by filtration) are stable in PBSwhile the particles without the BA are not. These data support theconclusion that the BA containing nanoparticles have the BA localized ontheir exterior as they are stabilized against aggregation in PBS.

Example 13 Transfection of MAP/pDNA Particles into HeLa Cells

HeLa cells were seeded at 20,000 cells/well in 24 well plates 48 h priorto transfection and grown in medium supplemented with 10% FBS. MAPparticles were formulated to contain 1 μg of pGL3 in 200 μL of Opti-MEMI at various charge ratios of polymer to pDNA (refer to Example 9).Growth medium was removed, cells washed with PBS and the particleformulation was added. The cells were subsequently incubated at 37° C.and 5% CO₂ for 5 h before the addition of 800 μL of growth mediumsupplemented with 10% FBS. After 48 h of incubation, a fraction of thecells were analyzed for cell viability using an MTS assay. The remainingcells were lysed in 100 μL of 1× Luciferase Cell Culture Lysis Reagent.Luciferase activity was determined by adding 100 μL of Luciferase AssayReagent to 10 ul of cell lysate and bioluminescence was quantified usinga Monolight luminometer. Luciferase activity is subsequently reported asrelative light units (RLU) per 10,000 cells. Results are shown in FIG. 8and FIG. 9.

Example 14 Co-Transfection of MAP/pDNA and/or siRNA Particles into HeLaCells

HeLa cells were seeded at 20,000 cells/well in 24 well plates 48 h priorto transfection and grown in medium supplemented with 10% FBS. MAPparticles were formulated to contain 1 μg of pGL3 and 50 nM of siGL3 in200 μL of Opti-MEM I at a charge ratio of 5+/−. Particles containingonly pGL3 or pGL3 and siCON were used as controls. Growth medium wasremoved, cells washed with PBS and the particle formulation was added.The cells were subsequently incubated at 37° C. and 5% CO₂ for 5 hbefore the addition of 800 μL of growth medium supplemented with 10%FBS. After 48 h of incubation, the cells were assayed for Luciferaseactivity and cell viability as described in Example 12. Results areshown in FIG. 10. Since the RLU is lowered in transfections with thesiGL3 (correct sequence), both the siGL3 and the pGL3 must beco-delivered.

Example 15 Transfection of MAP/siGL3 into HeLa-LUC Cells

HeLa-LUC cells (contain gene encoding for the firefly luciferaseprotein) were seeded at 20,000 cells/well in 24 well plates 48 h priorto transfection and grown in medium supplemented with 10% FBS. MAPparticles were formulated to contain 50 and 100 nM siGL3 in 200 μL ofOpti-MEM I at a charge ratio of 5+/−. Growth medium was removed, cellswere washed with PBS and the particle formulation was added. The cellswere subsequently incubated at 37° C. and 5% CO₂ for 5 h before theaddition of 800 μL of growth medium supplemented with 10% FBS. After 48h of incubation, the cells were assayed for Luciferace activity and cellviability as described in Example 12. Results are shown in FIG. 11.Since the RLUs decline with increasing concentration of siGL3, thesedata suggest that inhibition of an endogenous gene can occur.

Example 16 Synthesis of Mucic Acid Diiodide, (10)

1 g (2.7 mmol) of mucic acid diamine (Example 3) was mixed with 3.8 mL(27.4 mmol) of triethylamine and 50 mL of anhydrous DMF prior to thedropwise addition of 1.2 mL (13.7 mmol) iodoacetylchloride in a 250 mLround bottom flask. This mixture was allowed to react overnight underconstant stirring at room temperature. The solvent was subsequentlyremoved by vacuum pump, the product filtered, washed with methanol anddried under vacuum to yield 0.8 g (1.3 mmol, 46%) of mucic aciddiiodide. ¹H NMR ((CD₃)₂SO) δ 8.20 (s 2H), 2H), 7.77 (s, 2H), 4.11 (m,2H), 4.03 (m, 2H), 3.79 (m, 2H), 3.11-3.17 (m, 2H), 1.78 (d, 2H). ESI/MS(m/z): 652.8 [M+Na]⁺

Example 17 Synthesis of Mucic Acid Dicysteine, (11)

To 7 mL of 0.1 M degassed sodium carbonate was added 17 mg of L-cysteineand 0.4 g of mucic acid diiodide. The resulting suspension was broughtto reflux at 150° C. for 5 h until the solution turned clear. Thismixture was then cooled to room temperature and adjusted to pH 3 via 1 NHCl. Slow addition of acetone was then employed for productprecipitation. After filtration, washing with acetone and vacuum drying,60 mg of crude product was obtained.

Example 17 Synthesis of Mucic Acid Dicysteine, (11)

To 20 mL of 0.1 M degassed sodium phosphate buffer at pH 7.5 in a 50 mLround bottom flask was added 0.38 g of L-cysteine (3.2 mmol) and 0.40 g(0.6 mmol) of mucic acid diiodine. The resulting suspension was allowedto reflux at 75° C. overnight, cooled to room temperature andlyophilized. 80 mL of DMF was subsequently added to this lyophilizedlight brown powder and separation of the insoluble excess reagent andphosphate salts from the soluble product was achieved by filtration. DMFwas removed under reduced pressure and the product was vacuum dried togive 12 mg (0.02 mmol, 3%) of mucic acid dicysteine.

Example 18 Polymer Synthesis, (Poly(Mucic Acid-DiCys-PEG)) (12)

12 mg (21.7 μmol) of mucic acid dicysteine and 74 mg (21.7 μmol) ofPEG-DiSPA 3400 were dried under vacuum prior to the addition of 0.6 mLof anhydrous DMSO under argon in a 2 neck 10 mL round bottom flask.After 10 min of stirring, 9 μL (65.1 μmol) of anhydrous DIEA wastransferred to the reaction vessel under argon. This mixture was stirredunder argon overnight. The polymer containing solution was then dialyzedusing a 10 kDa membrane centrifugal device and lyophilized to yield 47mg (58%) of Poly(Mucic Acid-DiCys-PEG).

This polymer containing polyols is an anionic AB polymer.

Example 19 Covalent Attachment of Drug (Camptothecin, CPT) to Mucic AcidPolymer, (13)

10 mg (2.7 μmol of repeat units) of Poly(Mucic Acid-DiCys-PEG) wasdissolved in 1.5 mL of anhydrous DMSO in a glass jar. After stirring for10 min, 1.1 μL of DIEA (6.3 μmol), 3.3 mg (6.3 μmol) of TFA-Gly-CPT, 1.6mg (8.1 μmol) of EDC and 0.7 mg (5.9 μmol) of NHS were added to thereaction mixture. After stirring for 8 hrs, 1.5 mL of ethanol was addedand the solvents were removed under reduced pressure. The precipitatewas dissolved in water and insoluble materials were removed byfiltration through a 0.2 μm filter. The polymer solution was thendialyzed against water via a 10 kDa membrane and subsequentlylyophilized to give the Poly(Mucic Acid-DiCys-PEG)-CPT conjugate.

Example 20 Formulation of Nanoparticle with CPT-Mucic Acid Polymer (13)in Water, (20)

The Effective diameters of poly(Mucic Acid-DiCys-PEG) and poly(MucicAcid-DiCys-PEG)-CPT conjugate were measured by formulating the polymersin double distilled water (0.1-10 mg/mL) and evaluated via dynamic lightscattering (DLS) using a ZetaPALS (Brookhaven Instrument Co) Instrument.3 successive runs of 1 min each were subsequently recorded and averaged.The zeta potentials of both compounds was measured in a 1.1 mM KClsolution using a ZetaPALS (Brookhaven Instrument Co) Instrument. 10successive automated runs at target residuals of 0.012 were thenperformed and results averaged (FIG. 14). In particular, there twodistributions were measured for the poly(Mucic Acid-DiCys-PEG)-CPTconjugate the predominant distribution was a 57 nm (60% of the totalparticle population). A second minor distribution was also was measuredat 233 nm.

Example 21 Formulation of Boronic Acid-PEGylated Nanoparticle withCPT-Mucic Acid Polymer (13) and Boronic Acid-Disulfide-PEG₅₀₀₀ (6) inWater

The boronic acid PEGylated poly(Mucic Acid-DiCys-PEG)-CPT nanoparticleis formulated by dissolving the polymer in double distilled water at aconcentration of 0.1 mg/mL followed by the addition of Polymer 6(BA-PEG) also in water, such that the ratio of BA-PEG to the diols onthe mucic acid sugar in the poly(Mucic Acid-DiCys-PEG)-CPT conjugate is1:1. The mixture is incubated for 30 mins after which the effectivediameter and zeta potential are measured using a ZetaPALS (BrookhavenInstrument Co) instrument.

Example 22 Targeted Nanoparticles for pDNA Delivery in Mice

The plasmid pApoE-HCRLuc contains the gene to express luciferase and isunder the control of a liver specific promoter. Polymer (MAP) 4 (0.73mg), polymer 6 (73 mg) and polymer 9 (0.073 mg) were combined in 5 mL ofwater and then 1.2 mL of water containing the pApoE-HCRLuc plasmid wereadded (gives a charge ratio of polymer 4 to the plasmid of +3). Theparticles were placed in D5W (5% glucose in water) by successive spinfiltering with subsequent additions of D5W (starting from the initialformulation that was in water). Nude mice were implanted with Hepa-1-6liver cancer cells and tumors were allowed to grow until a size ofapproximately 200 mm³. Injections of the targeted nanoparticles weredone i.v. in the tail vein at an amount equal to 5 mg plasmid/kg mouse.The mice were imaged 24 hours after the injections. The mice showed nosigns of toxicity and there was luciferase expression detected in theregion of the tumor and not in the region of the liver.

The examples set forth above are provided to give those of ordinaryskill in the art a complete disclosure and description of how to makeand use the embodiments of the particles, compositions, systems andmethods of the disclosure, and are not intended to limit the scope ofwhat the inventors regard as their disclosure. Modifications of theabove-described modes for carrying out the disclosure that are obviousto persons of skill in the art are intended to be within the scope ofthe following claims. All patents and publications mentioned in thespecification are indicative of the levels of skill of those skilled inthe art to which the disclosure pertains. All references cited in thisdisclosure are incorporated by reference to the same extent as if eachreference had been incorporated by reference in its entiretyindividually.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books, orother disclosures) in the Background, Summary, Detailed Description, andExamples is hereby incorporated herein by reference.

It is to be understood that the disclosures are not limited toparticular compositions or biological systems, which can, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting. As used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the content clearly dictates otherwise. The term “plurality”includes two or more referents unless the content clearly dictatesotherwise. Unless defined otherwise, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which the disclosure pertains.

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice for testing of the specificexamples of appropriate materials and methods are described herein.

A number of embodiments of the disclosure have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the presentdisclosure. Accordingly, other embodiments are within the scope of thefollowing claims.

REFERENCES

-   Davis Mark E., Chen Zhuo (Georgia) and. Shin Dong M “Nanoparticle    therapeutics: an emerging treatment modality for cancer” in Nature    2008 vol. 7, pages 771-782-   Duncan Ruth “Polymer conjugates as anticancer nanomedicines” Nature    2006 vol. 6, pages 688-701-   Allen Theresa. M “Ligand-Targeted Therapeutics In Anticancer    Therapy” in Nature 2002 vol. 2 pages 750-763-   Liu Yemin and Reineke Theresa M. “Hydroxyl Stereochemistry and Amine    Number within Poly(glycoamidoamine)s Affect Intracellular DNA    Delivery” in J. Am. Chem. Soc. 2005, 127, 3004-3015

What is claimed is:
 1. A nanoparticle comprising a polymer containing apolyol, a polymer containing a boronic acid, and one or morepolynucleotides, wherein the polymer containing a boronic acid iscoupled to the polymer containing a polyol with a reversible covalentlinkage, and wherein the nanoparticle is configured to present thepolymer containing a boronic acid to an environment external to thenanoparticle.
 2. The nanoparticle of claim 1, wherein at least one ofthe polynucleotide encodes a protein.
 3. The nanoparticle of claim 2,wherein at least one of the polynucleotide is DNA or RNA.
 4. Thenanoparticle of claim 3, wherein the RNA is mRNA.
 5. The nanoparticle ofclaim 1, wherein at least one of the polynucleotide is siRNA, shRNA, ormiRNA.
 6. The nanoparticle of claim 1, wherein at least one of thepolynucleotide is an oligonucleotide.
 7. The nanoparticle of claim 1,wherein at least one of the polynucleotide is an aptamer.
 8. Thenanoparticle of claim 1, wherein said polynucleotide comprises one ormore of DNA, RNA, mRNA, siRNA, shRNA, miRNA, an oligonucleotide, or anucleic acid aptamer.
 9. The nanoparticle of claim 1, wherein thepolymer containing a polyol comprises one or more of at least one of thefollowing structural units

wherein A is an organic moiety of formula

in which R₁ and R₂ are independently selected from any carbon-based ororganic group with a molecular weight of 10 kDa or less; X isindependently selected from an aliphatic group containing one or more of—H, —F, —C, —N or —O; and Y is independently selected from —OH or anorganic moiety presenting an —OH, and B is an organic moiety linking oneof the R₁ and R₂ of a first said moiety A with one of the R₁ and R₂ of asecond said moiety A in the polymer.
 10. The nanoparticle of claim 9,wherein R₁ and R₂ independently have the formula:

wherein d is from 0 to 100; e is from 0 to 100; f is from 0 to 100; Z isa covalent bond linking one organic moiety to another, and Z₁ isindependently selected from —NH₂, —OH, —SH, and —COOH.
 11. Thenanoparticle of claim 9, wherein X is C_(n)H_(2n+1), in which n is to0-5; and wherein Y is —OH.
 12. The nanoparticle of claim 9, wherein A isindependently selected from the group consisting of

wherein the spacer is independently selected from any organic group; theamino acid is selected from any organic group bearing a free amine and afree carboxylic acid group; n is 1-20; and Z₁ is independently selectedfrom —NH₂, —OH, —SH, and —COOH.
 13. The nanoparticle of claim 12,wherein A is independently selected from


14. The nanoparticle of claim 9, wherein B is

in which q is 1-20; p is 20-200; and L is a leaving group.
 15. Thenanoparticle of claim 9, wherein the structural unit of formula (I) is:


16. The nanoparticle of claim 9, wherein the structural unit of formula(II) is:


17. The nanoparticle of claim 9, wherein the structural unit of formula(III) is:

in which n is 1-20.
 18. The nanoparticle of claim 1, wherein the polymercontaining a polyol is


19. The nanoparticle of claim 1, wherein the polymer containing aboronic acid comprises at least one terminal boronic acid group and hasthe general formula:

wherein R₃ and R₄ are independently an hydrophilic organic polymer, X₁is an organic moiety containing one or more of —C, —N, or —B, Y₁ is analkyl group of formula —C_(m)H_(2m)—, in which m is ≧1 or an aromaticgroup, r is 1-1000, a is 0-3, and b is 0-3.
 20. The nanoparticle ofclaim 19, wherein R₃ and R₄ are (CH₂CH₂O)_(t), where t is from 2 to2000.
 21. The nanoparticle of claim 19, wherein X₁ is —NH—C(═O)—, —S—S—,—C(═O)—NH—, —O—C(═O)— or —C(═O)—O— and Y₁ is a phenyl group.
 22. Thenanoparticle of claim 19, wherein r=1, a=0 and b=1.
 23. The nanoparticleof claim 19, wherein functional group 1 and functional group 2 are thesame or different and are independently selected from —B(OH)₂, —OCH₃,—OH.
 24. The nanoparticle of claim 19, wherein the polymer containing aboronic acid is:

wherein s=20-300.
 25. The nanoparticle of claim 24, wherein the polymercontaining a polyol is


26. The nanoparticle of claim 25, wherein said polynucleotide comprisesone or more of DNA, RNA, mRNA, siRNA, shRNA, miRNA, an oligonucleotide,or a nucleic acid aptamer.
 27. The nanoparticle of claim 1, furthercomprising a targeting ligand.
 28. The nanoparticle of claim 27, whereinthe targeting ligand is a protein, a small molecule, an antibody, or anantibody fragment.
 29. The nanoparticle of claim 28, wherein thetargeting ligand is selected from transferrin, folic acid and galactose.30. A composition comprising the nanoparticle of claim 1 and a suitablevehicle and/or excipient.
 31. The composition of claim 30, wherein thecomposition is a pharmaceutical composition and the suitable vehicleand/or excipient is a pharmaceutically acceptable vehicle and/orexcipient.
 32. A method to deliver a polynucleotide to a target, themethod comprising contacting the target with the nanoparticle ofclaim
 1. 33. The method of claim 32, wherein the target is a cell withinthe body of a mammal.
 34. A system to deliver a polynucleotide to atarget, the system comprising the nanoparticle of claim 1 to be used todeliver the polynucleotide to the target.
 35. A method to administer apolynucleotide to an individual, the method comprising administering tothe individual the nanoparticle of claim 1 comprising at least onepolynucleotide.
 36. A system for administering a polynucleotide to anindividual, the system comprising the nanoparticle of claim 1 to beadministered to the individual.
 37. A method to prepare a nanoparticlecomprising a polymer containing a polyol, a polymer containing a boronicacid, and a polynucleotide the method comprising contacting the polymercontaining a polyol with at least one polynucleotide for a time andunder conditions to allow for particle formation contacting theparticles formed by the polymer containing a polyol and thepolynucleotide with a polymer containing a boronic acid for a time andunder conditions to allow coupling of the polymer containing a polyolwith the polymer containing a boronic acid.
 38. A nanoparticlecomprising a polymer containing a polyol, a polymer containing a boronicacid, and an antibody or antibody fragment, wherein the polymercontaining a boronic acid is coupled to the polymer containing a polyolwith a reversible covalent linkage, and wherein the nanoparticle isconfigured to present the polymer containing a boronic acid to anenvironment external to the nanoparticle.